This fellowship application seeks support for the dissertation research of an individual MD/PhD student and proposes a training plan that will equip her to become an independent physician-scientist at a major research institution. The research project aims to elucidate the biochemical regulation of cytosine oxidation, a newly- discovered epigenetic phenomenon that factors into diverse processes, including embryonic development, pluripotency, and malignancy. Among the four bases of DNA, cytosine provides the major substrate for chemical modifications that impact gene expression. The methylation of cytosine to form 5-methylcytosine (5mC) is well established as a critical mediator of gene silencing in cellular differentiation, imprinting, and genomic stability. Recently, however, ten-eleven translocation (TET) enzymes were found to oxidize 5mC into three additional bases-5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC)-that maintain a stable presence in mammalian genomes, suggesting they could have distinct epigenetic functions. The most important function discovered thus far is DNA demethylation, since the oxidized bases provide pathways to regenerate unmodified cytosine. The presence of the various cytosine modifications in the genome appears to be an intricate balancing act, which when perturbed contributes to numerous cancers, as evidenced by abnormal genomic patterns of DNA methylation that are increasingly studied as cancer signatures. Major questions remain regarding how TET enzymes regulate the balance and genomic distribution of these oxidized cytosines. The goal of this proposal is to shed light on these frontiers using a combination of biochemical approaches and innovative genome sequencing technologies. Aim 1 proposes to decipher how TET's substrate preferences help to control stepwise synthesis of the oxidized cytosines. TET enzymes preferentially act on CpG dinucleotides and have three possible substrates (5mC, 5hmC, and 5fC), which can be present on one or both complementary DNA strands. HPLC- and mass spectrometry-based assays will be used to quantify the reactivity of the three TET isoforms to all of their possible substrates. This would mark the first systematic study of TET's kinetic properties, dissected into individual oxidation steps for each of its substrates and isoforms, which would elucidate key elements of the biochemical regulation of cytosine oxidation. Aim 2 proposes a novel method of localizing 5fC and 5caC at single nucleotide resolution in the genome. The specific base excision enzyme thymine DNA glycosylase (TDG) will be exploited to cleave 5fC and 5caC from the genome, resulting in abasic sites that will be mapped by next-generation sequencing. This would represent a significant improvement over current methods and provide the first base-resolution, genome-wide map of 5caC. This method will be applied to primordial germ cells and their malignant counterpart, seminoma cells, which together with Aim 1 would provide important insights into the potential epigenetic functions of TET enzymes and oxidized cytosines in physiological and pathological settings.